1. Field of the Invention
This invention pertains to the field of in vitro sialylation of glycoproteins, including recombinant glycoproteins.
2. Background
The circulatory lifetime of glycoproteins in the blood is highly dependent on the composition and structure of its N-linked carbohydrate groups. This fact is of direct relevance for therapeutic glycoproteins which are intended to be administered parenterally. In general, maximal circulatory half life of a glycoprotein requires that its N-linked carbohydrate groups terminate in the sequence NeuAc-Gal-GlcNAc. Without the terminal sialic acid (NeuAc), the glycoprotein is rapidly cleared from the blood by a mechanism involving the recognition of the underlying N-acetylgalactosamine (GalNAc) or galactose (Gal) residues (Goochee et al. (1991) Bio/Technology 9: 1347–1355). For this reason, ensuring the presence of terminal sialic acid on N-linked carbohydrate groups of therapeutic glycoproteins is an important consideration for their commercial development.
In principle, mammalian cell culture systems used for production of most therapeutic glycoproteins have the capacity to produce glycoproteins with fully sialylated N-linked carbohydrate groups. In practice, however, optimal glycosylation is often difficult to achieve. Under the conditions of large scale production, overproduction of the glycoprotein by the cell can outstrip its ability to keep up with glycosylation, and this capability can be positively and negatively influenced by many subtle variables in culture conditions (Goochee et al., supra.).
Production of glycoproteins in transgenic animals has some of the same problems as mammalian cell culture. While the “production” of a glycoprotein is inherently better controlled it is also less susceptible to manipulation. If glycosylation is not complete, there is little that can be done with the animals to alter the outcome. With transgenic animals there is often another problem. While the predominant sialic acid in humans is N-acetyl-neuraminic acid (NeuAc), goats, sheep and cows all produce a large fraction of their total sialic acid as N-glycolyl-neuraminic acid (NeuGe). Although the impact of this modification is not yet fully explored from a functional or regulatory perspective, it is known that the NeuGc substitution is antigenic in humans (Varki (1992) Glycobiology 2: 25–40)
Since the most important problems associated with glycosylation of commercially important recombinant and transgenic glycoproteins, involve terminal sialic acid, a need exists for an in vitro procedure to enzymatically “cap” carbohydrate chains that lack a terminal sialic acid. With such a procedure, the problem encountered with transgenic glycoproteins could also be addressed by resialylation with NeuAc once the “non-human” sialic acid NeuGc was removed. The ideal method would employ a sialyltransferase that is capable of efficiently sialylating N-linked or 0-linked oligosaccharides of recombinant glycoproteins on a practical scale. The present invention fulfills these and other needs.